The present invention concerns a mutation responsible for autosomal prelingual non-syndromic deafness and a method for the detection of this hereditary sensory defect for homozygous and heterozygous individuals. The invention concerns more particularly a specific deletion of at least one nucleotide in the connexin 26 (Cx 26) gene and especially in a guanosine rich region, notably between the nucleotides 27 and 32. The invention is also directed to the use of polynucleotide, or fragments thereof, for example as tools useful for the in vitro detection of a mutation of a gene belonging to the Cx26 gene family.
Profound or severe prelingual deafness affects one child in a thousand in developed countries (Morton N E. Genetic epidemiology of hearing impairment. In Genetics of hearing impairment. (The New York Acad Sci, New York 1991; 630:16-31). It is a major handicap as it impedes language acquisition.
According to studies performed in a U.S. population of children with non-syndromic (isolated) prelingual deafness and in whom an obvious environmental cause has been excluded, it is estimated that up to two-thirds of the cases have a genetic basis (Marazita M L, Ploughman L M, Rawlings B, Remington E, Arnos K S, Nance W E. Genetic epidemiological studies of early-onset deafness in the U.S. school-age population. Am J Med Genet 1993; 46:486-91). These forms are mainly sensorineural and are almost exclusively monogenic. The major mode of inheritance is autosomal recessive (DFMB), involving 72% to 85% of cases, this fraction increasing to 90% when only profound deafness is taken into account.
Autosomal recessive prelingual deafness is known to be genetically highly heterogeneous. Estimates of the number of DFNB loci vary from thirty to one hundred (Petit C. Autosomal recessive non-syndromal hearing loss. In Genetics and Hearing Impairment. Martini A, Read A P, Stephens D, eds (Whurr, London) 1996; 197-212), for a review), of which fourteen have so far been mapped to the human chromosomes (Petit C. Genes responsible for human hereditary deafness: symphony of a thousand. Nature Genet 1996; 14:385-91) for review, (verhoeven K, Van Camp G, Govaerts P J, et al. A gene for autosomal dominant non-syndromic hearing loss (DFNAl2) maps to chromosome 11q22-24. Am J Hum Genet 1997; 60:1168-74 and Campbell D A, McHale D P, Brown K A, et al. A new locus for non-syndromal autosomal recessive sensorineural hearing loss (DFNB16) maps to human chromosome 15q21-q22. J Med Genet 1997; in press).
A majority of the families attending genetic counseling clinics consist of normal hearing parents with a single deaf child who wish to know the risk of recurrence of the defect. In most cases, given the major role of environmental causes of prelingual deafness, it is not usually possible even to recognize whether the hearing loss is of genetic origin. Genetic counseling in such families would be greatly improved by an ability to detect DFNB mutations. In this respect, the high genetic heterogeneity of the condition represents a major obstacle.
After the initial identification of the DFNB1 locus on 13q11 in a large consanguineous Tunisian family (Guilford P, Ben Arab S, Blanchard S, et al. A non-syndromic form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q. Nature Genet 1994; 6:24-8), two studies performed on New Zealand/Australian families (Maw MA, Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population. Am J Hum Genet 1995; 57:629-35), and on Italian/Spanish families (Gasparini P, Estivill X, Volpini V, et al. Linkage of DFNB1 to non-syndromic neurosensory autosomal-recessive deafness in Mediterranean families. Eur J Hum Genet 1997; 5:83-8) suggested that this locus might be a major contributor to prelingual deafness in these populations, although individual lod scores obtained in these families were not significant owing to the small size of these families.
Recently, the Cx26 gene, which: encodes a gap junction protein, connexin 26, has been shown to underlie DFNB1 deafness. Two different C- greater than A substitutions resulting in premature stop codons in three DFNB1 linked consanguineous Pakistani families have been reported (Kelsell D P, Dunlop J, Stevens H P, et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 1997; 387:80-3). These two substitutions were identified, respectively, at codon 77 and at codon 24. This result has offered the opportunity directly to assess this hypothesis.
The difficulties encountered in genetic counseling for prelingual non-syndromic deafness due to the inability to distinguish genetic and non-genetic deafness in the families presenting a single deaf child was one of the reasons that led the inventors to undertake a characterization of the spectrum and prevalence of mutations present in the Cx26 gene in 35 families from several parts of the world with autosomal recessive prelingual deafness.
The determination of a mutation in the Cx26 gene has notably rendered possible the use of a detection probe as a tool for the identification of a specific form of autosomal prelingual non-syndromic deafness, and more particularly the useful role of a newly identified 30delG (a G deletion at position 30; position 1 being the first base of the initiator. codon) mutation in such families. This invention establishes that the contribution of the DFNB1 locus predominantly results essentially from the 30delG mutation. It is now believed that the 30delG accounts for about three-quarters of all recessive DFNB1 mutations.
The invention is thus intended to provide a purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which in a wild form encodes a polypeptide implicated in hereditary sensory defect. The mutated purified polynucleotide presents a mutation responsible for prelingual non-syndromic deafness.
The invention also provides oligonucleotides comprising of 15 to 50 consecutive nucleotides of the mutated purified polynucleotide that are useful as primers or as probes.
In addition, the invention aims to supply a method and a kit for the detection of the hereditary sensory defect for homozygous as heterozygous individuals.
According to the invention, the purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which encodes in a wild form a polypeptide implicated in hereditary sensory defect, presents a mutation responsible for prelingual non-syndromic deafness selected from the group consisting of a specific deletion of at least one nucleotide.
By mutation, according to the invention it means a specific deletion of at least one nucleotide. Thus, a mutated sequence means a polynucleotide sequence comprising at least a mutation.
A chain of nucleotides, according to the invention, means a polynucleotide, which encodes not necessarily a polypeptide, but which presents between 27 and 2311 nucleotides linked together.
The invention particularly concerns a purified polynucleotide wherein, the specific mutation is a deletion located in a region encoding connexin 26 of chromosome 13q11-12, preferably located in a guanosine rich region starting at nucleotide 27 preferably at nucleotide 30, and extending to nucleotide 32 or nucleotide 35, all the recited nucleotides being inclusive. More particularly according to the invention, the specific deleted purified polynucleotide encodes for a truncated polypeptide.
By truncated polypeptide, according to the invention it means a fragment of the polypeptide, which does not present the properties of the wild form of the polypeptide either in length, in amino acid composition, or in functional properties.
A preferred embodiment of a specific deletion is a guanosine deletion at position 30, also called xe2x80x9c30delG mutationxe2x80x9d. Another preferred embodiment of the specific deletion is a 38 bp deletion beginning at position 30.
The invention also includes a purified polynucleotide, which hybridizes specifically with any one of the polynucleotides as defined above under the following stringent conditions: at low temperatures between 23xc2x0 C. and 37xc2x0 C., in the presence of 4xc3x97SSC buffer, 5xc3x97Denhardt""s solution, 0.05% SDS, and 100 xcexcg/ml of salmon sperm DNA. (1xc3x97SSC corresponds to 0.15 M NaCl and 0.05M sodium citrate; 1xc3x97Denhardt""s solution corresponds to 0.02% Ficoll, 0.02% polyvirylpyrrolidone and 0.02% bovine serum albumin)
The invention also concerns an oligonucleotide useful as a primer or as a probe comprising 15 to 50 consecutive nucleotides of the polynucleotide according to any one of the polynucleotides as defined above. The oligonucleotide sequence is selected from the following group:
A first couple:
5xe2x80x2-TCTTTTCCAGAGCAAACCGCC(SEQ ID No. 1)-3xe2x80x2
5xe2x80x2-TGAGCACGGGTTGCCTCATC(SEQ ID No. 2)-3xe2x80x2.
The length of the PCR product has been obtained from 285 bp in length;
A second couple allowing to explore the other part of the reading frame:
5xe2x80x2-GACACGAAGATCAGCTGCAG(SEQ ID No. 3)-3xe2x80x2
5xe2x80x2-CCAGGCTGCAAGAACGTGTG(SEQ ID No. 4)-3xe2x80x2
A third couple:
5xe2x80x2-CTAGTGATTCCTGTGTTGTGTGC(SEQ ID No. 9)-3xe2x80x2; and
5xe2x80x2 ATAATGCGAAAAATGAAGAGGA(SEQ :ID No. 10)-3xe2x80x2 and
A fourth couple:
5xe2x80x2-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCTAGTGATTCCT GTGTTGTGTGC(SEQ ID No. 14)-3xe2x80x2; and
5xe2x80x2 ATAATGCGAAAAATGAAGAGGA(SEQ ID No. 10)-3xe2x80x2.
Another oligonucleotide useful as a probe is selected from the following group:
5xe2x80x2-AGACGATCCTGGGGGTGTGAACAAA(SEQ ID No. 5)-3xe2x80x2
5xe2x80x2-ATCCTGGGGGTGTGA(SEQ ID No. 6)-3xe2x80x2
5xe2x80x2-AGACGATCCTGGGGGCTCACCGTCCTC(SEQ ID No. 7)-3xe2x80x2.
In addition, the invention concerns a method for the detection of an hereditary sensory: defect, namely autosomal prelingual non-syndromic deafness, for homozygous as heterozygous individuals in a biological sample containing DNA, comprising the steps of:
a) bringing the biological sample into contact with a oligonucleotide primers as defined above, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample;
b) amplifying the DNA;
c) revealing the amplification products;
d) detecting the mutation.
Step d) of the above-described method may comprise a Single-Strand Conformation Polymorphism (SSCP), a Denaturing Gradient Gel Electrophoresis (DGGE) sequencing (Smith, L. M., Sanders, J. Z., Kaiser, R. J., Fluorescence detection in automated DNA sequence analysis. Nature 1986; 321:674-9); a molecular hybridization capture probe or a temperature gradient gel electrophoresis (TGGE).
Step c) of the above-described method may comprise the detection of the amplified products with an oligonucleotide probe as defined above.
According to the invention, a biological sample can be a blood sample extracted from people suffering from any kind of deafness with any criteria as follows: neurosensorial or mixed isolated deafness, advanced or not, at any degree of severity, concerning familial or sporadic case, or individuals exposed to noise, or individuals suffering from a low acoustic, or individuals susceptible to carry an anomaly in the gene, or from an embryo for antenatal diagnostic.
Another aim of the invention comprises a method for the detection of an hereditary sensory defect, the autosomal prelingual non-syndromic deafness, for homozygous and heterozygous individuals in a biological sample containing DNA, comprising the steps of:
a) bringing the biological sample into contact with an oligonucleotide probe according to the invention, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample; and
b) detecting the hybrid formed between the oligonucleotide probe and the DNA contained in the biological sample.
Step b) of the above-described method may consist in a single-strand conformation. Polymorphism (SSCP), a denaturing gradient gel electrophoresis (DGGE) or amplification and sequencing.
The invention also includes a kit for the detection of an hereditary sensory defect, the autosomal prelingual non-syndromic deafness, for homozygous as heterozygous individuals, said kit comprising:
a) oligonucleotides according to the invention;
b) the reagents necessary for carrying out DNA amplification; and
c) a component that makes it possible to determine the length of the amplified fragments or to detect a mutation.